Abstract- TMAO and Incident Subclinical CVD, Clinical CVD, and Mortality Trimethylamine-N-oxide (TMAO) is an intestinal microbiome-dependent metabolite of L-carnitine, a nutrient in red meat, chicken, and fish, and lecithin, a nutrient in eggs, milk, meats and fish. In in vitro and animal models, TMAO promotes atherosclerosis, independently of traditional cardiovascular disease (CVD) risk factors. Thus, TMAO represents a novel molecule that may account for links between diet and CVD and provide a clinically relevant molecular target for novel interventions to reduce risk. Yet, the effects of TMAO on CVD risk in humans are not well established, with mixed evidence from a limited number of prior studies. This research will provide critical evidence on how TMAO relates to initiation, extent, and onset of atherosclerosis and clinical CVD in two independent, large, well-established, prospective community-based US cohort studies: the Multi-Ethnic Study of Atherosclerosis (MESA) and Cardiovascular Health Study (CHS). MESA allows prospective investigation of onset of subclinical atherosclerosis (coronary artery calcium, CAC); and CHS, of clinical CVD events. This will provide robust, complementary evidence on how TMAO may influence atherosclerotic risk. The large number of health behaviors, risk factors, and subclinical and clinical disease outcomes in these cohorts will also facilitate multivariable adjustment as well as analyses related to other subclinical CVD outcomes. The inclusion of two separate, large, prospective community-based cohorts of well- phenotyped US adults, including both middle-aged and older adults of diverse races/ethnicities with existing standardized measures of sociodemographics, diet, lifestyle, CVD risk factors, coronary calcium, subclinical CVD, and incident CVD events, will provide the most complete picture of how TMAO relates to initiation, extent, and clinical onset of CVD, of the independence of these relationships from other sociodemographic and lifestyle factors, and of possible heterogeneity in these relationships. The results will have tremendous impact for understanding whether and how TMAO influences CVD, directly informing the need for and design of large intervention studies to reduce TMAO levels including novel lifestyle strategies and novel pharmacologic and molecular interventions targeting both TMAO and the microbiome in humans.